Tuberculosis (TB) is the leading infectious killer of youth and adults and the first most common infectious disease worldwide. One third of the world's population is currently infected and 20 million of those infected are active cases; TB will kill 30 million people this decade. More than 50 million people may already be infected with multidrug-resistant (MDR) strains of TB. Prior to MDR tuberculosis, the success rate of drug combination treatment was greater than 90%, even in AIDS patients. MDR tuberculosis, however, is not only highly infectious but also essentially incurable with a mortality of 50%.
Tuberculosis is caused by infection with Mycobacterium tuberculosis, a bacillus bacterium. It is spread by aerosol droplets and causes irreversible lung destruction. Recently, because of complications due to multidrug-resistant strains, the number and combination of antibiotics administered must be individually tailored depending on the strain the patient is harboring. In general, manifest disease with an MDR strain of Mycobacterium tuberculosis—a strain resistant to both isoniazid and rifampin, and possibly to additional drugs—has a poor clinical outcome since efficient therapeutic strategies are still lacking.
Initially, antimicrobial susceptibility testing of Mycobacterium tuberculosis is carried out with a primary set of drugs, consisting of the front-line drugs isoniazid, rifampin, ethambutol, pyrazinamide, and, optionally, streptomycin. If resistance to one or several of these drugs is detected, it is common practice to test an extended spectrum of antimicrobial compounds.
For quite some time three different growth-based laboratory methods have been accepted for determining antimicrobial susceptibility of Mycobacterium tuberculosis: (1) the resistance ratio method, (2) the absolute concentration method, and (3) the proportion method. Most laboratories in the Western hemisphere utilize a modified proportion method on solid medium. For most of the major antituberculous agents, this technique defines resistance of Mycobacterium tuberculosis as a percentage of resistant organisms larger than 1 percent in a given population of bacilli.
Because antimicrobial susceptibility testing on solid media requires visible growth of the organisms (which requires three weeks of incubation), testing is preferentially done in liquid media today.
In the last decade antimicrobial susceptibility testing has become a dynamic field spawning many new technologies. They all comply with the standard set by the Centers for Disease Control and Prevention that susceptibility testing results for Mycobacterium tuberculosis have to be available within 28 days of the time the specimen arrives in the laboratory (Bird B R. et al, J Clin Microbiol 996;34:554-559.).
An increasing number of approaches assess drug susceptibility by identifying alternative markers of drug-resistant metabolic activities. Among those are colorimetry, flow cytometry (Norden, M A. et al, J Clin Microbiol 1995; 33:1231-1237), bioluminescence assay of mycobacterial adenosine triphosphate (Nilsson, L E et al, Antimicrob Agents Chemother 1988; 32:1208-1212.), and quantitation of mycobacterial antigens (Drowart, A. et al., Int J Tuberc Lung Dis 1997; 1:284-288.). Mycobacteriophage-based methods, for example, with luciferase reporter phages or PhaB phages, appear to be promising as well (Jacobs, W Jr et al., Science 1993; 260:819-822). However, the complexities of these technologies and high cost have largely hampered their wider application in the clinical mycobacteriology laboratory.
Molecular biology as a tool to detect resistant TB. Mycobacterium tuberculosis resistance to drugs always results from mutations. These mutations are either deleterious for the bacterial cell or, conversely, alter the structure of a protein targeted by a drug without compromising the protein's function for the microorganism. In Mycobacterium tuberculosis these mutations appear to be confined to chromosomal DNA and do not involve mobile genetic elements (such as plasmids).
In particular, DNA sequencing, but also other techniques such as gel electrophoresis (single-stranded conformation polymorphism [SSCP]-PCR, dideoxy fingerprinting) and hybridization on solid phase (line probe assay, DNA chip technology) or on liquid phase (heteroduplex analysis, mismatch cleaving assay, molecular beacon) can identify those subtle mutations.
Resistance to rifampin, the most important component of current treatment regimens, is associated with a short core region consisting of 27 amino acids in the rpoB gene, which codes for the β subunit of RNA polymerase (Telenti, A. et al, Lancet 1993; 341:647-650). The ethambutol resistance-determining region (ERDR) has been proposed as a mutational hot spot in the embB gene, whereas the situation with pyrazinamide resistance is less clear. Resistance to isoniazid appears to be the complex result of single or multiple mutations in the katG, inhA, oxyR-ahpC, and/or kasA gene(s) (Heym, B. et al, Lancet 1994; 344:293-298.). Similarly, mutations in the rpsL and/or rrs gene(s) correlate with resistance in approximately 80 percent of streptomycin-resistant strains (Böttger, E C. Trends Microbiol 1994; 2:416-421).
In light of the worsening global TB epidemic and the extreme vulnerability of HIV-infected individuals to TB, rapid and reliable antimicrobial susceptibility testing in the laboratory is paramount for proper management of patients, particularly those with MDR TB.
Given the above, current available assay cannot quickly and completely detect drug-resistant Mycobacterium tuberculosis. It requires a quick assay with high specificity and sensitivity to detect drug-resistant Mycobacterium tuberculosis from available samples, especially from sputum of suspected patients.